Method of identifying a biologically-active composition from a biofilm

ABSTRACT

The subject invention provides materials and methods that effectively support innate immunity and/or disperse pathogenic biofilms using readily available, nontoxic, natural substances, while supporting restoration of normal microbiotic homeostasis. In one embodiment, the subject invention provides anti-biofilm compositions comprising one or more probiotic organisms, anti-microbial honey, and other ingredients such as prebiotic compounds, other hive products, green tea derivatives, other plant derivatives, and vitamin D3.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation of co-pending U.S. application Ser.No. 16/699,383, filed Nov. 29, 2019; which is a continuation of U.S.application Ser. No. 16/105,017, filed Aug. 20, 2018, which is acontinuation application of U.S. application Ser. No. 15/626,971, filedJun. 19, 2017, now U.S. Pat. No. 10,086,025, which is a continuation ofU.S. application Ser. No. 15/484,842, filed Apr. 11, 2017, now U.S. Pat.No. 10,004,771, which is a continuation of U.S. application Ser. No.15/412,712, filed Jan. 23, 2017, now U.S. Pat. No. 9,919,012, which is acontinuation of U.S. application Ser. No. 15/349,371, filed Nov. 11,2016, now U.S. Pat. No. 9,717,765, and U.S. Ser. No. 15/349,420, filedNov. 11, 2016, now U.S. Pat. No. 9,713,631; which are continuationapplications from U.S. application Ser. No. 13/503,325, filed May 31,2012; now U.S. Pat. No. 9,504,739; which is a National Stage Applicationof International Application Number PCT/US2011/059370; filed Nov. 4,2011; which claims the benefit of U.S. provisional application Ser. No.61/447,735, filed Mar. 1, 2011, all of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

In a world where only the fittest survive, bacteria have a remarkablecapacity to adapt and evolve in response to their environment. Somemicrobes have evolved a critical survival advantage through theirsymbiotic relationship with their host. In exchange for a convenientgrowth “niche” in and on their human hosts, these bacteria confercertain important survival advantages, such as direct inhibition ofpathogen colonization on the surfaces where these “friendly” bacteriathrive. Under ordinary circumstances the human immune system would havefought off these selfsame bacteria as hostile invaders. But byconferring certain health benefits upon their human hosts, these“probiotic” bacteria create a beneficially synergistic, evolutionarilyadvantaged and thus evolutionarily conserved relationship betweenmicrobe and man.

Among the benefits conferred by probiotic organisms is stimulation ofthe host innate immune system. As the main and first defender againstall infections, the integrity, efficiency and rapidity of the innateimmune system response is critical for host survival. An intact andsmoothly functioning innate immune system protects the host as well asits probiotic organisms by limiting host pathogen colonization. Whilebeneficial to the host, such protection is also self-serving to theprobiotic organism, since direct inhibition of pathogen growthconversely also promotes the growth of healthy, beneficial organisms onthese same surfaces. Therefore, these benefits just as importantlyprotect the microbiota conferring this advantage while simultaneouslyprotecting the host itself. Most microbes, however, are not probiotic,but are at best, nonpathogenic, and at worst, possibly lethal.

The century-old germ theory—that only one free-floating “germ” ormicrobe is needed to confer infection—directly shaped the subsequentstudy of pathogens and their resulting infections. Antibiotics, whichare the main tools in treating infections, are based on the efficiencyof microbial killing of microbes studied in free-floating (planktonic)state, functioning as a single cell. Quantification of antibioticefficacy is done in traditional Minimum Inhibitory Concentration (MIC)assays. Traditional microbiology has been wrong, however. To thecontrary, most human infections are now understood to be due to thecoordinated, en masse behavior of entire microbial colonies. Thesecolonies are composed of microbes working together to secrete anextracellular matrix called biofilm which surrounds and protects theentire colony from antibiotics and attack by an intact immune system.

Biofilms are initiated when free-floating, planktonic bacteria anchor tobiologic or inert surfaces such as indwelling medical devices. Theattached bacteria multiply and progress from a state of monolayer to amicrocolony and then to a critical mass, at which bacterial crosstalkoccurs, triggering a phenomenon known as quorum sensing that leads tothe biofilm phenotype. Quorum sensing turns on biofilm-producing genesnot expressed or produced in non-sessile bacteria. The bacteria respondcollectively to express factors that are specific to the biofilmphenotype, which lead to the secretion of an exopolysaccharide (EPS)matrix definitive of biofilm. This biofilm phenotype is characterizedmorphologically by the formation of microbial towers, which are composedof layers of embedded, live bacteria with intervening water channels.Under the right environmental conditions, free-floating bacteria arereleased from the biofilms, and the cycle is continued at othersurfaces.

Approximately 80% of the world's microbial biomass resides in thebiofilm state, and the National Institutes of Health estimates that morethan 75% of microbial infections that occur in the human body areunderpinned by the formation and persistence of biofilms. Suchinfections include dental caries, periodontitis, musculoskeletalinfections, osteomyelitis, bacterial prostatitis, endocarditis, chronicbronchitis and other states of chronic lower respiratory inflammation,cystic fibrosis pneumonia, otitis media, chronic tonsillitis,adenoiditis and device infections.

Although it might seem that biofilms are “just another type ofinfection”, pathogenic biofilms behave completely differently than thevery same bacteria in free-floating, non-biofilm producing form. Due tocompletely different genomic expression, biofilm related infections havea different clinical course and antibiotic response than planktonic-typeinfections. Moreover, treating biofilm associated infections “the same”as planktonic infections creates antibiotic-resistant “superbugs”because the EPS matrix generated by the colony gives the colony1000-fold resistance against antibiotics which would ordinarily killthese microbes if in free-floating form.

Because antibiotics fail to eradicate these EPS-protected microbialcommunities, use of antibiotics actually compounds the problem becauseantibiotics select for and perpetuate increasingly antibiotic-resistantbacteria. These “super bugs” include methicillin resistantStaphylococcus aureus (MRSA), the world's leading cause of nosocomialinfection, and a bacterium now widespread in the community at large.Despite the global ramifications of inadvertent “super bug” creation,modern medicine has few treatments for pathogenic biofilm associatedinfections. Furthermore, the solution to this problem is not merely thedevelopment of another new antibiotic, because in order to avoidperpetuation of antibiotic-resistant “super bugs”, such treatments musthave broad-spectrum as well as anti-biofilm activity. This is reflectedtime and time again in real patients, for whom even repeat, extendedcourses of antibiotics “proven” effective in MIC tests are oftenunsuccessful.

Attacking, dissolving or otherwise weakening the bacterial biofilmmatrix, interrupting the quorum mechanisms maintaining the bacterialcommunity, as well as upregulating local host innate immunity could curewhat would otherwise become incurable chronic infection or chronicbiofilm-associated inflammatory disease. Penetration or dispersion ofthe bacterial biofilm “armor” is critical in fighting biofilm-inducedchronic inflammation, particularly those involving “super bugs”.

In vitro antibiotic efficacy test results can dramatically underestimatethe protection conferred by pathogenic biofilms in vivo against thetested and supposedly effective antibiotic. Due to biofilm's protectiveproperties, antibiotic choices based on these results may be irrelevant,misleading and even clinically harmful. Indeed, even repeat, extendedcourses of antibiotics demonstrated in MIC studies as effective areoften unsuccessful in patients afflicted with biofilm-associatedinflammatory states.

Not only are bacteria in biofilm state robustly resistant toantibiotics, they are also resistant to other anti-bacterials andbiocides, such as alcohols, acids and iodine solutions. In fact, today's“antiseptics” such as popular hand “sanitizers” may be part of theproblem, since use of such biocides may actually increase the prevalenceof pathologic biofilms on involved surfaces, such as the hands ofhealthcare workers. Therefore, developing non-antibiotic methods ofinducing biofilm dissociation and/or prevention of biofilm secretion isan area of increasing research.

Not all biofilm is pathogenic, however. Gastrointestinal probioticssecrete biofilm that protects the mucosal surface against intestinalinfection by pathogenic biofilm-forming organisms. Additionally,synergistic probiotics, such as certain species of Lactobacillus and E.coli, give the host other benefits, such as normal intestinal motility,toxin elimination and the efficient absorption of nutrients such asvitamin B12. Moreover, from an evolutionary perspective, the human bodyrequires colonization by probiotic microbes for survival advantage. Thismutual interdependence exemplifies the synergistic relationship betweenhuman host and its beneficial microbiota.

The human host mounts an inflammatory reaction as a normal response topathogen invasion and accompanying biofilm formation. If such aninflammatory reaction is sustained, this inappropriate over-stimulationof an initially normal immune response can result in damage to anddisease of the human host itself. However, probiotic organisms, unliketheir pathogenic counterparts, maintain a healthy and balanced immuneresponse. In other words, probiotics maintain homeostasis between hostinflammatory and anti-inflammatory reactions.

Inflammation is a complex phenomenon, involving recruitment of whiteblood cells, leakage of fluid from capillaries as well as release ofchemical mediators and oxidants necessary to kill invading microbes.Maintaining immune homeostasis is important because host-producedinflammation can cause damage to the host itself. These very sameprocesses, if not “switched off” once eradication of pathogens hasoccurred, result in local tissue damage, bodily harm and consequentdisease.

Probiotic organisms upregulate the body's immune surveillance againstpathogens but also down-regulate inflammatory signals. This constantbalancing act by probiotics helps to maintain the delicate but criticalhomeostasis between immune stimulation and immune over-stimulation.Disruption of this homeostasis can result in certain common humandiseases with a common unifier of chronic inflammatory state such asmeibomian gland dysfunction and chronic rhinosinusitis.

Biofilms have broad-ranging clinical relevance in all areas of medicine.Bacterial biofilms such as those commonly associated with Pseudomonasand Staphylococcus are known to be a cause of intractable infection aswell as chronic low-grade inflammation. They consist of colonies ofbacterial organisms that collectively secrete and form a protectivelayer of extracellular matrix material. The bacterial colonies inbacterial biofilms appear to be very resistant to the hosts' naturaldefenses as well as antibiotic treatments. Biofilms colonize virtuallyany surface in or on the human body to which these colonies can adhere.They often colonize biomaterials such as urinary catheters,transcutaneous intravenous lines and prosthetic heart valves.

Dry eye is a medical condition that affects >10 million people in theUnited States alone. It is arguably the most common ophthalmiccondition. Its frequency in general ophthalmology practices can be ashigh as 50%. It results from the deficiency in the production and/orcomposition of tears produced by the eye's lacrimal and adnexalsecretory glands. The eye depends on the constant flow of tears tolubricate the surface of the eye, maintaining vision and overall comfortof the eyes. Tears are composed of water, oils, mucus, antibodies andother proteins. These are all normally secreted by the lacrimal glandlocated around the eyes and the meibomian glands of the eye lids. Whenthere is an imbalance in the amount of tears and/or abnormalities in thecomposition and/or amount of the tear constituents, a person mayexperience many different symptoms of dry eye—blurring of the vision,eye irritation, redness, itching, pain and sensation of ocular “foreignbody”.

The vast majority of dry eye conditions are due to meibomian glanddysfunction, associated with ocular rosacea, blepharitis and ocularallergy. Repercussions of dry eye syndrome include significant negativeimpact on quality of life, corneal damage, ongoing ocular and periocularinflammation and even infection. Common symptoms of dry eye syndromeinclude dry, scratchy, sandy or gritty feeling, foreign body sensation,pain or soreness, stinging, burning, eye fatigue, itch, increased blinkfrequency, photophobia, blurry vision, redness, mucus discharge,intolerance of contact lens wear and even excessive tearing. Thesesymptoms can be due to many conditions, including lupus, rheumatoidarthritis, Sjogren's syndrome, normal aging, contact lens use, anycorneal surgery such as LASIK, diabetes, meibomian gland dysfunction ofany cause, anatomic abnormality, extended computer use, and medications,as well as other common ocular surface disorders such as allergicconjunctivitis. Increased leukocytes and cytokine mediators are found onthe ocular surface of the dry eye, indicating ongoing inflammation.

Lid and ocular hygiene methods are commonly recommended in an attempt todilute and remove local irritants and inflammatory chemicals thought tobe influencing chronic ocular and periocular inflammation. The mostcommon recommendations include dilute baby shampoo lid scrubs as well asother over-the-counter cleansers. However, none of these products issufficiently antibacterial to kill eyelid bacteria within clinicallyrelevant exposure times.

Although dry eye syndrome has many causes, the pathology common to dryeye syndrome regardless of cause is abnormal change on the ocularsurface due to alterations in quality or quantity of tears. Tear fluidconsists of 3 layers—a hydrophilic mucus layer and aqueous and lipidlayer. Adjacent to the cornea, the mucus layer is produced byconjunctival goblet cells and absorbed by corneal surface glycoproteins.Despite potentially normal quantity of tear production, deficiency ordysfunction of the mucin itself can lead to poor wetting and/orglycation of the corneal surface, and thus desiccation and epithelialdamage common in dry eye syndrome. Forming the majority of tear volume,the aqueous layer is secreted by the lacrimal glands and is adjacent tothe mucus layer. The high volume and diffusability of the aqueouscomponent delivers nutrients and oxygen to the cornea, which does nototherwise itself receive a great amount of blood and nutrient flow. Thefinal layer is the lipid layer, secreted by the meibomian, Zeiss andMoll glands of the lids. Lipids in this layer function as surfactantsand emollients by lowering the surface tension of the aqueous fluid,allowing efficient dispersal of tear fluid over the ocular surface, andslowing evaporation of the aqueous layer of tears. Because the lacrimaland meibomian glands have androgen receptors, low androgen status canresult in abnormality of the lipid layer, hastening tear evaporation andresulting in dry eyes.

Dry eye syndrome can further be characterized by the component of tearfluid most affected. Therefore, dry eye syndrome can be divided intolubricant deficiency dry eye, aqueous tear deficient dry eye andevaporative dry eye. Lubricant deficiency dry eye involves abnormalityof the tear mucin layer. The mucin layer can be disrupted by a number ofconditions, including allergic conjunctivitis, direct chemicalirritation (such as preservatives in ocular drops), volatile mucosalirritants, viral infection, thermal damage, and nutritional/metabolicdisorders such as vitamin deficiency and protein malnutrition.

Aqueous tear deficient dry eye is due to abnormal function or amount ofthe aqueous layer secreted by the lacrimal gland. Tear deficiency canresult from many systemic conditions, such as Sjogren's syndrome,Sjogren's disease, lupus, rheumatoid arthritis, and diabetes, as well asthe normal aging process associated with lacrimal gland atrophy. Othercauses include ocular chemical irritants, lacrimal gland damage, viralinfection, menopause and medications such as diuretics, antihistamines,oral contraceptives or hormone therapy, anti hypertensives,antidepressants and systemic vasoconstrictors.

Evaporative dry eye is due to abnormality of the lipid layer. Becausethe lipid layer is unable to function as an effective surfactant andemollient, it causes excessive evaporation of the tear fluid layer. Mostcommonly, evaporative dry eye is due to meibomian gland dysfunction,environmental conditions (airborne irritants, low ambient humidity, highambient temperature) and computer use, which markedly lowers normalblink frequency, causing more rapid evaporation of tear fluid from thecorneal surface.

In addition to abnormality of the tear fluid layers, there are othercauses of dry eyes. These include anatomic (excessive exposure ofsurface of the eyes as in Grave's disease, eversion of eye lidsassociated with normal senescence) and neural causes. Neural stimulationof the ocular surface results normally in direct feedback to thelacrimal gland, which then adjusts secretion appropriately in response.This neural feedback is inhibited by peripheral nerve damage affectingocular sensation, cerebrovascular accident or, more commonly, LASIKcorneal surgery. Ocular and orbital surgery can cause dry eye syndromesimply due to the physical impact on the tissues with instrumentationand surgical trauma. Furthermore, abnormal proportions and/or amounts ofessential fatty acids (EFA) such as linoleic acid and imbalance betweenomega-3 and omega-6 EFAs can lead to ocular surface inflammation and dryeyes. Extended use of contact lenses can cause dry eyes due tomechanical interference with normal distribution of nutrients andoxygen, as well as the chronic deposition of matter that typicallyoccurs on contact lenses themselves. These micro-concretions become anidus for bacterial growth and pathogenic biofilm production fromPseudomonas and Staphylococcus as well as a cause of ongoingmicro-trauma to the corneal epithelium.

Regardless of the particular cause of ocular surface disturbance,chronic inflammation at the ocular surface is the end result. The innateimmune system appears to be the predominant initiator of this process.In dry eye syndrome of any cause, low-level, ongoing ocular surface andperi-ocular infiltration of immune cells such as conjunctival CD4 Tcells and corneal CD11b+ monocytes develops. Localized tissue stressinduced by ocular surface dryness induces secretion of inflammatorycytokines such as IL-1, TNF-alpha and IL-6. These substances activatenearby antigen presenting cells (APCs), which in turn cause theexpansion of Th17 cells producing IL-17 as well as Th1 cells producingIFN-gamma. The elaborated cytokines IL-17 and IFN-gamma perpetuate theinflammatory response by increasing leukocyte migration to the ocularsurface. Over time, this low-level inflammation can make the eye moresusceptible to bacterial, viral and other infections.

Although the high frequency of chronic dry eye syndrome in the generalpopulation establishes ongoing need, there are currently no particularlyeffective treatments for this condition. Over-the-counter treatmentsprovide short-lived symptomatic relief and fail to address theunderlying issue of ocular inflammation. Indeed, use of these productsoften aggravates dry eyes themselves. Ocular preservatives in artificialtears often worsen dry eye syndrome due to corneal damage resulting fromprolonged exposure to these chemicals. Overuse of topicalvasoconstrictors that “get the red out” also can exacerbate cornealinflammation. In the prescription drug category, in the past decade,only 1 new product going beyond the category of artificial tears hasbeen approved in the United States for treatment of dry eyes—namely,Restasis (cyclosporine). Topical cyclosporine is very costly, and has asignificant side effect profile, as well as a poor clinical responserate of only 15%. Therefore, relieving the symptoms of dry, irritatedand/or inflamed eyes is currently limited to ocular fluidsupplementation (i.e., use of artificial tears), surgical treatment viapunctal plug to decrease tear fluid loss into the nasolacrimal duct, useof potentially hazardous pharmaceutical drugs or therapeutic ocularequipment such as “moisture chamber spectacles” and therapeutic contactlenses. However, these methods are costly, unwieldy, can furtherperpetuate ocular pathology and moreover are only partially effective.

Current drug development is focusing on several new pharmaceuticalproducts. There is some evidence that topical hormonal therapy may helprelieve chronic dry eyes. Also in the pipeline are a modifiedcyclosporine, topical steroidal and non-steroidal anti-inflammatories,oral a-3 adenosine receptor agonists, synthetic anti-inflammatorymolecules known as resolvins, anti-LFA-1 compounds, pro-inflammatoryinterleukin antagonists, immunosuppressant monoclonal antibodies,topical antibiotics, chemical secretagogues and an artificial tearsolution containing hyaluronic acid. However, all of these with theexception of the last are expensive pharmaceutical products, and thehyaluronic acid drops function similarly to artificial tears already onthe market. Secondary treatment modalities have included topicalantibiotic therapy to address the often low-grade infections associatedwith dry eyes. Despite the frequency of dry eyes, universally acceptedtreatment modalities are inadequate, as is evidenced by currentextensive research and ongoing drug development in this area. Today'sresearch efforts are focusing on development of syntheticimmunomodulatory pharmaceuticals—no new over-the-counter ocularanti-inflammatory compound currently exists or is known to be planned.

Lactobacillus extracts have been previously used in cosmeticapplications (US Patent Application Number 11/70/810, L'Oreal patent,WO9907332, JP 3112983, JP 2002037739). However, Lactobacillus has notpreviously been used in the treatment of dry eyes. Honey has also beenused in cosmetic applications for the treatment of chronic skininflammation and/or infection. However, honey has not previously beenused in the treatment of dry eyes.

Prior to the subject invention, treatment of chronic sinusitis ofteninvolves pronged and repeated antibiotics, intranasal as well assystemic corticosteroids and even otolaryngologic surgery. However, eventhough chronic rhinosinusitis is increasingly recognized as abiofilm-related disease, no treatment exists which is directed at thebiofilm component of the condition itself. The same kind of solutionsproposed in this invention for dry eye syndrome can also be applied inthe treatment of and symptom relief from chronic rhinosinusitis.

Like chronic sinusitis, chronic periodontitis is widespread in thegeneral population. Along with genetic and environmental factors, dentalplaque biofilm is necessary for the development of chronic periodontaldisease. Even though there is inadequate evidence to establish causalityat this time, many studies have shown a clear and parallel relationshipbetween oral disease and atherogenesis in heart disease. Nevertheless,treatment of oral disease leads to both a reduction in the systemicinflammatory burden as reflected in inflammatory markers such as hsCRPand an improvement in endothelial function. Currently, however, the onlytreatments available for chronic gingival and periodontal disease aredebridement and antibiotics taken systemically or applied subgingivally.However, as stated above, antibiotics are generally poorly effectiveagainst pathogenic biofilms.

Other widespread chronic inflammatory disorders involve the respiratorytract. These include allergic rhinoconjunctivitis, chronic bronchitisand asthma. Less common conditions such as cystic fibrosis andaspergillosis have clearly been established to involve biofilms such asPseudomonas and Aspergillus biofilm, causing significant morbidity andmortality. Treatments in all of these conditions include steroids andsystemic antibiotics and antifungals. In particular, sometimes macrolideantibiotics, which are antibiotics with immunomodulatory properties, maybenefit patients with respiratory diseases associated with chronicinflammation, in part because they may decrease biofilm formation.However, there is no other non-invasive therapeutic option besidesantimicrobials or steroids at this time.

Today's antibiotics clearly and repeatedly demonstrate profound failureto treat biofilm-associated infection. Moreover, there are no well knownor proven anti-biofilm treatments per se. Attempts to treat infectionspresumed secondary to pathogenic biofilm formation include repeated andprolonged antibiotic therapy, physical removal of the biofilm (i.e.,surgery or debridement) and topical sterilizers such as alcohol basedfoams or gels used for hand cleansing. Not only do these treatments failto restore normal physiology, they disrupt the homeostasis of innateimmunity—antibiotics breed increasingly resistant “super bugs”, surgeryor debridement results in anatomic wounding which creates anotherpotential site for infection, and topical disinfectants may encouragedevelopment and growth of pathogenic biofilms by eradicating normalcommensals as well as pathogens.

It is likely that current anti-infectives may be failing to treat manyof the world's infections because such treatment fails to treat thebiofilm component, and in fact, may even result in increased pathogenicbiofilm growth and worsened infection. Moreover, formation andattachment of the biofilm itself may create the window of opportunityenabling that particular pathogen to cause infection. In order to beeffective, treatments must be targeted against pathogen biofilmdisruption, must support rather than disturb normal innate immunity andshould interrupt quorum sensing mechanisms responsible for maintainingpathogenic biofilms.

In the ocular field, it would be very useful if it were possible toelicit anti-inflammatory and/or anti-biofilm effects directly on theocular surface using safe, inexpensive and locally administeredcompounds, rather than antibiotics, synthetic immunomodulators orsystemically delivered compounds. It would also be very useful to beable to further limit or even avoid the use of chemical preservativespresent in most ocular drops, as these chemicals themselves can worsenocular surface inflammation.

Presently, there is no method or treatment available for elicitinganti-inflammatory and anti-biofilm effects on the ocular surface ofindividuals with symptoms of dry eye syndrome, dry eyes and/orchronically inflamed, red or irritated eyes. Moreover, there is aprofound lack of non-antibiotic, non-invasive anti-biofilm therapy whichcan potentially cure antibiotic-resistant “superbug” infections withoutperpetuating the global problem of antibiotic resistance. Furthermore,current treatments such as antibiotics or surgical intervention havesignificant associated side effects, cost, and treatment failure as wellas repercussions on the rest of the body, including the “uninvolved”areas.

It would also be desirable for a treatment to be applied directly to theareas affected by pathogenic biofilms, including surfaces such as humanmucosa and keratinized and non-keratinized epithelium. Such topicaladministration techniques would circumvent systemic toxicity, since theyare by definition administered via localized (skin medicament, nasalspray, oral inhaler or nebulizer, ocular drop, oral troche, et cetera)delivery systems. Also desirable would be for treatments to beinexpensive and safe, for example, if treatments were to be comprised ofnatural, generally regarded as safe (GRAS) derivative/non-pharmaceuticalingredients. Lastly, it would be useful if anti-biofilm compounds couldbe applied to inert surfaces (i.e., hospital equipment, airplane traytables, school desks) to limit the spread/presence of pathogenicbiofilms in the hospital/clinical environment as well as in thecommunity at large.

SUMMARY OF THE INVENTION

The subject invention provides materials and methods that effectivelysupport innate immunity and/or disperse pathogenic biofilms usingreadily available, nontoxic, natural substances, while supportingrestoration of normal microbiotic homeostasis.

The compositions of the subject invention can be delivered to theaffected tissues by direct application, significantly increasing bothefficacy and safety. Because the composition is applied directly to anarea affected by a pathogenic biofilm, including surfaces such as humanmucosa and keratinized and non-keratinized epithelium, it directlyaddresses the current need for non-invasive anti-biofilm treatments.

Examples of such locally directed therapies include skin medicaments,nasal sprays and washes, ear drops, oral inhalers and nebulizers, oculardrops, contact lenses, contact lens solutions, oral troches, dentifricessuch as mouthwash, toothpaste, floss, periodontal treatment, etc. Ineach case, the composition of the present invention is administered viaa vehicle whose composition is physiologically appropriate based on thearea of anatomic administration.

Also based on anatomic area of involvement, the present invention mayuse a two or more step application process, e.g., localized applicationof a first composition to decrease pathological biofilms, followed byapplication of a second composition to promote restoration of normalcommensal bacterial homeostasis.

The compositions of this invention can also be applied to inert surfaces(e.g. hospital equipment, airplane tray tables, school desks) to limitthe spread/presence of pathogenic biofilms in the environment as well asin the community at large.

Another advantage of the current invention is the established safety ofits components. The compositions described herein are composed ofcomponents that have already been individually established to be safe.In preferred embodiments, the compositions of the subject inventioncomprise a mixture of natural, generally regarded as safe (GRAS)ingredients.

Yet another advantage of the current invention is its potential to becombined with antibiotics. Since the invention has anti-biofilm effect,it makes the underlying biofilm associated infection susceptible toantibiotics typically ineffective in the biofilm treatment setting. Theinvention also allows antibiotics to be used at a lower amount, therebydecreasing toxicity as well as treatment expense, since the invention“sensitizes” the underlying pathogenic micro-organisms to antibioticantimicrobial mechanism(s).

Some ingredients common to many, but not all, embodiments of thecompositions of this invention include microbial metabolites, cellularand/or acellular fractions used singularly or in combination with viableor nonviable probiotic or other microbes, including bacteria, fungi andcyanobacteria such as Arthrospira (Spirulina) platensis, andpharmaceutical grade honey. Other ingredients that may be used incertain embodiments include, but are not limited to, prebiotic compoundssuch as larch or acacia gum, other hive products such as royal jelly,bee bread and propolis, green tea derivatives such as epigallocatechingallate (EGCG) and L-theanine, other plant derivatives such as fromInula helenium, Melaleuca alternifolia and Leptospermum scoparium andwater-soluble and water-insoluble Vitamin D3.

Advantageously, in preferred embodiments, ingredients of the compositionof the current invention work together to inhibit biofilm-associatedinfections while improving associated chronic inflammatory conditionsthrough enhancement of pathogenic biofilm dispersion as well asimprovement of the normal, local innate immune response.

In the area of ocular and adnexal tissue application, the compositionsof the current invention can be used for the treatment of underlyinginflammatory processes associated with dry eye syndrome. The sequelae ofpathogenic biofilms on or near the ocular surface can result in chronicocular low-grade inflammatory conditions, including dry eye syndrome.The subject invention provides compositions for treating the symptomsand the causes of dry eye syndrome. Specifically, these compositionsinhibit pathogenic biofilm growth and bring about an overallanti-inflammatory effect on the ocular/adnexal surface.

Such topical treatment of the ocular and adjoining surfaces improves thehomeostasis between pathogenic and beneficial microflora of theocular-adnexal area. Rebalancing or adjusting pathogenic versusnonpathogenic or even beneficial organisms improves symptoms ofchronically dry, irritated, red or inflamed eyes. Such an improvementcan be brought about by an embodiment of the invention comprising atopically applied mixture of live or dead micro-organisms, and/or theirextracts, as well as pharmaceutical grade honey that possessesanti-biofilm effect. Additionally, other compounds such as L-theanine,Vitamin D3, prebiotic polysaccharides, and the marine organism Spirulinacan be used according to the subject invention to treat conditionsassociated with pathological biofilm.

The subject invention is based in part on the recognition of the role ofbiofilms in chronic ocular inflammatory states, specifically, dry eyesyndrome. The function of the ocular and adnexal microbiome is to“boost” the local innate immune system and protect the colonizedsurface. Cross talk between the commensal microbial flora and ocularmucosal and immune epithelial cells helps maintain ocular surfacehomeostasis and ocular surface health. Commensals colonizing the ocularsurface include such diverse micro-organisms as Staphylococci,Corynebacterium, Streptococcus and Proprionibacterium. This microbiomeremains relatively stable unless disturbed. However, there are manycommon situations which are likely to affect healthy ocular andperi-ocular microbiome balance—antibiotics and other medications,contact lenses, blepharitis, meibomian gland dysfunction, ocular rosaceaor other causes of chronically irritated and/or dry eyes. When normalocular and peri-ocular micro-organism populations are disturbed by anynumber of possible, common causes, ocular surface irritation,inflammation and discomfort result.

Application topically applied mixture as described herein results indecreased inflammation of the ocular surface and surrounding areas.Since the many disparate causes of dry eye disease are united by thesame immunopathogenesis of chronic inflammation, the invention may beused by the general public at large for symptomatic improvement ofchronically dry, red, irritated and/or inflamed eyes.

Beyond treatment of dry eye, the compositions of the current inventioncan be used for the prevention and/or disruption of pathologicalbiofilms and/or chronic infections present in, associated with, orleading to, various other chronic inflammatory states such as chronicrhinosinusitis; chronic periodontitis; chronic bronchitis and otherstates of respiratory inflammation including aspergillosis, cysticfibrosis and asthma; inflammatory otic conditions such as “swimmer'sear,” otitis externa and chronic otitis; and inflammatory skinconditions such as atopic dermatitis and eczema. The pathophysiology ofthese conditions is likely to involve the disruption of the normalcommensal bacterial population by pathogenic species and pathogenicbiofilm formation. The subject invention improves symptoms associatedwith these conditions and the underlying inflammatory state.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides materials and methods that effectivelysupport innate immunity and/or disperse pathogenic biofilms usingreadily available, nontoxic, natural substances, while supportingrestoration of normal microbiotic homeostasis.

Elements common to many but not all embodiments of the invention includea combination of one or more probiotic organisms, such as those of thegenus Lactobacillus acidophilus, Saccharomyces cerevisiae orEscherischia coli, a fraction thereof and/or metabolite thereof, incombination with an anti-microbial honey with anti-biofilm effect, suchas manuka honey (Medi-Honey, Comvita, New Zealand or manuka honeysterilized via ISO 11137-2a 2006 validating 15 kGy radiationsterilization dose for health care products, Food Technology ServiceIncorporated, Mulberry, Fla.).

Other elements that may be used in other, but not necessarily all,embodiments include prebiotic compounds such as larch or acacia gum,other hive products such as royal jelly, bee bread and propolis, greentea derivatives such as epigallocatechin gallate (EGCG) and L-theanine,and other plant derivatives such as from Inula helenium and the manukabush, as well as Vitamin D3.

In its most general form, the invention provides a compositioncomprising a mixture of active components, which may itself be added toany vehicle appropriate for the site of application.

This particular embodiment of the invention is independent of thevehicle or carrier used in its administration. For instance, such anembodiment may be used as an “ingredient,” which can be added to otherexisting or yet-to-be developed products. One formula for this“ingredient” form of this invention includes pharmaceutical grade honeyand a probiotic organism. For instance:

100 B CFU lyophilized *S. thermophilus+100 μL honey=1 B CFU/ml 100%honey  Formula 1:

250 μL 100 B CFU/ml *S. thermophilus+500 μL honey=25 B CFU/ml 75%honey  Formula 2:

500 μL 100 B CFU/ml *S. thermophilus+500 μL honey=50 B CFU/ml 50%honey  Formula 3:

750 μL 100 B CFU/ml *S. thermophilus+250 μL honey=75 B CFU/ml 33%honey  Formula 4:

900 μL 100 B CFU/ml *S. thermophilus+100 μL honey=90 B CFU/ml 10%honey  Formula 5:

*Specific strain S. thermophilus ATCC BAA-250; however, other strainsmay be substituted.

Gamma irradiated honey such as Medi-Honey (Comvita Inc., New Zealand) orfilter sterilized honey may be used undiluted or may be diluted insterile normal saline solution to varying final concentrations prior tothe addition of probiotic. An organism such as freeze-dried S.thermophilus (NCFB 2393 or LMG 18311 or ATCC BAA-250) may be used andadded to 100% honey or the honey-saline solution as above to obtain afinal concentration of 100 Billion CFU/ml of the mixture.

Another formula for this “ingredient” form of the invention includes anenriched source of biosurfactant of 1 mg or more derived from probioticorganism(s). The yield varies with organism strain type and cultureconditions. This enriched biosurfactant fraction can be diluted instandard PBS solution for varying biosurfactant concentrations. Forinstance:

1000 μg **S. thermophilus biosurfactant in 1000 μL ***PBS for 1 mg/mldilution  Formula 6:

500 μg **S. thermophilus biosurfactant in 1000 μL ***PBS for 0.5 mg/mldilution  Formula 7:

500 μg **S. thermophilus biosurfactant in 250 μL ***PBS for 2 mg/mldilution  Formula 8:

500 μg **S. thermophilus biosurfactant in 100 μL ***PBS for 5 mg/mldilution  Formula 9:

10 mg **S. thermophilus biosurfactant in 1 mL ***PBS for 10 mg/mldilution  Formula 10:

*Embodiments of biosurfactant isolation methods are described below.**Specific strain S. thermophilus ATCC BAA-250; however, other strainsmay be substituted.***Honey such as Medi-Honey or anti-biofilm characterized honey may besubstituted for sterile, standard solution of PBS. Either such solutionmay be further diluted as needed.

The compositions of the subject invention can be applied directly to theinvolved areas, such as human mucosal, keratinized and non-keratinizedepithelial surfaces. This technique reduces or eliminates systemictoxicity, because the administration is localized (skin medicament,nasal spray, oral inhaler or nebulizer, ocular drop, oral troche, etcetera).

Additionally, based on anatomic area of involvement, the presentinvention may use a two or more step application process, i.e.,localized application of a first formulation to decrease pathologicalbiofilms, followed by application of a second formulation to assist inrestoration of normal commensal bacterial homeostasis.

Various embodiments of the invention include ocular drops, gel,ointment, cream or other vehicle of delivery of the active compoundsappropriate to area of application, periocular lotion, gel, ointment,cream or other vehicle of delivery appropriate to the area ofapplication, intranasal aqueous or non-aqueous spray, nasal salinerinse, skin soap, lotion, cream, emollient, and solution such as meantfor contact lens cleaning and maintenance or spray.

Anti-biofilm efficacy of constituents, extracts, and/or mixtures of thepresent invention may be assessed using the Calgary Biofilm Device, anFDA Class I approved device for the inoculation of biofilms (U.S. Pat.No. 6,599,714, herein incorporated by reference) to perform the MBEC(Minimum Biofilm Eradication Concentration) procedure or other means ofassessing anti-biofilm efficacy. This may include a method described indetail below. Other anti-microbial tests that can be employed include:the agar or disk-diffusion technique, the Kirby-Bauer test and theMinimum Inhibitory Concentration (MIC). These techniques are well knownto those versed in the state of the arts and will not be recounted indetail here. Protocols may be found in “Techniques in Microbiology” byJohn Lammert, Pearson Education, 2007, and “Microbiology LaboratoryFundamentals and Applications” by George A. Wistreich, PearsonEducation, 2003, which are incorporated by reference in their entirety.

Current high-throughput anti-biofilm efficacy testing is limited toessentially one test, namely the MBEC test (see above, U.S. Pat. No.6,599,714). This particular test has a number of limitations, includingnon-reusability and expense of the test plates as well as the lack ofeffective shear force around each individual peg upon which biofilms aresupposed to grow. Without adequate shear force, strong biofilm formationis not stimulated to occur and true in vivo biofilm conditions are notlikely to have been approximated.

However, microbial biofilms may be grown on relatively non-porous media,such as spheres, in a rotating-tube reactor system. This non-porousmedia may be represented by sterile 5 mm glass spheres. The reactorvessel may be a sterile 50 ml tube polystyrene Falcon tube or a similarcolumn placed on a rotating shaker. The tube or column may havewater-tight tops such that rotations on a mechanical rotator do notresult in spillage of vessel contents (i.e., contents are secured viascrew-top, rubber or cork plug). The spheres may be immobilized at oneend of the tube or column by an open mesh, autoclavable plastic plug.Each tube or column may contain sufficient open space such that as thedevice is mechanically rotated, a flushing action is generated toprovide shear force to the cellular organisms adhering to the spheresurfaces without generating vacuum or static effect upon the mediawithin the enclosed system. Devices such as tube inlets and tube outletscirculated via filtration pumps permit media replacement and asepticremoval of individual spheres by use of sterile forceps or similardevice. Circulating biological growth media may be sterilized viamembrane filtration. Positive determination of adequate biofilmformation growing on spheres may be performed via various microscopicstaining techniques.

The biofilm which has grown on the spheres may be tested against anarray of antimicrobials placed in antimicrobial susceptibility wells.Well plates or similar devices are used to create small, standard agarlawns. This technique is well known to those well versed in the state ofthe art and will not be recounted in detail here. Protocols may be foundin “Techniques in Microbiology” by John Lammert, Pearson Education,2007, and “Microbiology Laboratory Fundamentals and Applications” byGeorge A. Wistreich, Pearson Education, 2003, which are incorporated byreference in their entirety. Agar is prepared, autoclaved and aliquotedinto small vessels, admixed with varying concentrations of testantimicrobials and allowed to cool. Upon solidification of the agar,circular wells are punched from the agar lawn in a size equivalent tothe size of the biofilm sphere. The sphere is then placed in the well.

The plates containing the wells and biofilm-coated spheres are coveredand incubated in a humidified incubator at conditions appropriate forthe particular organism being studied. Degree of anti-biofilm efficacyis represented by direct visualization of bacterial growth around theparticular well, i.e., the smaller the diameter of growth, the greaterthe anti-biofilm efficacy of that particular compound. Alternatively,degree of anti-biofilm efficacy may be measured via color change createdby conversion of certain stains admixed into the agar medium to enhancevisualization of biofilm growth.

Antibiofilm efficacy (Biofilm Inhibitory Concentration or BIC) can becompared directly against planktonic efficacy by performing the MinimumInhibitory Concentration (MIC) test for the same anti-microbialcompounds and micro-organisms being tested. Additionally, antibiofilmefficacy can be measured using a classification system similar to themanuka factor (Molan, Peter, “Method for the assay of antibacterialactivity of honey”, 2005, herein incorporated by reference), exceptthat, in this case, what is measured is the size of complete biofilmgrowth inhibition (biofilm inhibitory concentration, or BIC), ratherthan the killing diameter (“zone of inhibition”) of antimicrobialsubstances of compounds such as honey. This procedure will be used todevelop BIC standards of individual compounds, including honey, againsta range of bacteria, as well as complexes of compounds together, againstbacterial groups such as gram negative bacteria, methicillin sensitiveand methicillin resistant Staphylococcus, et cetera.

In another high-throughput variation of the invention, biofilm-coatedspheres as described above may be placed directly into 96 well platesand subjected to a large battery of antimicrobial compounds and/orconcentrations. Biofilms may be removed from these spheres viasonication and the resulting supernatants interpreted via both platingand spectrophotometry in a process similar to that described in the MBECprocedure (see previous reference).

During culture of any organism for use in the invention, cultures may ormay not be grown to maximal plateau growth phase at which time they maybe harvested for maximal biofilm production.

In certain embodiments, cellular or acellular fractions or extracts oforganisms or their extracellular milieu such as a biofilm derivativeitself may have particular anti-biofilm and/or anti-inflammatoryefficacy that may be even more effective than the source of the fractionitself.

Biofilm Inhibitory Compositions of the Subject Invention

The ingredients of the subject invention can be prepared in a variety offorms including, but not limited to, powders, suspensions, andsolutions. In addition, various ingredients, such as probiotics, can beprepared as lyophilized powders or culture supernatant and/or, whereappropriate, in a concentrated form.

In certain embodiments, the therapeutic composition comprises at leastone probiotic organism at a concentration (B CFU/weight of thecomposition) of at least about 0.001 B CFU/g, 0.005 B CFU/g, 0.01 BCFU/g, 0.05 B CFU/g, 0.1 B CFU/g, 0.5 B CFU/g, 1 B CFU/g, 5 B CFU/g, 10B CFU/g, 50 B CFU/g, 100 B CFU/g, or 500 B CFU/g.

In certain embodiments, the therapeutic composition comprises at leastone probiotic organism at a concentration (B CFU/weight of thecomposition) of no greater than about 0.05 B CFU/g, 0.1 B CFU/g, 0.5 BCFU/g, 1 B CFU/g, 5 B CFU/g, 10 B CFU/g, 50 B CFU/g, 100 B CFU/g, 500 BCFU/g, or 1000 B CFU/g.

In certain embodiments, the therapeutic composition comprises aprobiotic microorganism at a concentration (B CFU/weight of thecomposition) ranging from about 0.001 B CFU/g to 100 B CFU/g, 0.1 BCFU/g to 90 B CFU/g, 5 B CFU/g to 80 B CFU/g, 10 B CFU/g to 70 B CFU/g,or 30 B CFU/g to 50 B CFU/g.

In certain embodiments, the therapeutic composition comprises aningredient at a concentration (weight of the ingredient/weight of thecomposition) of at least about 1 μg/g, 5 μg/g, 10 μg/g, 20 μg/g, 50μg/g, 0.1 mg/g, 0.5 mg/g, 1 mg/g, 5 mg/g, 10 mg/g, 50 mg/g, 100 mg/g, or500 mg/g, wherein the ingredient is selected from the group consistingof extracts of microorganisms, chemical substituents, cellular oracellular components, and/or metabolites of probiotic microorganisms,honey, hive products, biosurfactants, prebiotics, plant extracts, andvitamin D.

In certain embodiments, the therapeutic composition comprises aningredient at a concentration (weight of the ingredient/weight of thecomposition) of no greater than about 10 μg/g, 50 μg/g, 0.1 mg/g, 0.5mg/g, 1 mg/g, 10 mg/g, 50 mg/g, 100 mg/g, or 500 mg/g, wherein theingredient is selected from the group consisting of extracts ofmicroorganisms, chemical substituents, cellular or acellular components,and/or metabolites of probiotic microorganisms, honey, biosurfactants,prebiotics, plant extracts, and vitamin D.

In certain embodiments, the ingredient is selected from probioticmicroorganisms, chemical substituents, cellular or acellular components,and/or metabolites of probiotic microorganisms.

In one embodiment, the therapeutic composition has a pH of above 5.0,5.5, 6.0, 6.3, 6.5, 6.7, or 7.0. In one embodiment, the therapeuticcomposition has a pH of below 9.0, 8.5, 8.0, 7.7, 7.5, 7.3, or 7.0.

Identifying Biofilm Inhibitory Activity

Advantageously, certain compositions of the subject invention canprevent or inhibit the formation of pathogenic biofilms. In addition,certain compositions of the subject invention can reduce, control oreliminate existing pathogenic biofilms.

The compositions of the subject invention can prevent or inhibit theformation of pathogenic biofilms, and/or reduce, control or eliminateexisting pathogenic biofilms via a variety of mechanisms, includingpreventing, inhibiting, and/or disrupting the deposition, adhesion,and/or anchoring of biofilms or pathogenic microorganisms to biologicalor non-biological surfaces; preventing, inhibiting, and/or disruptingthe secretion and/or release of extracellular factors such asexopolysaccharide (EPS) matrix; and/or preventing, inhibiting, and/ordisrupting quorum-sensing mechanisms.

In one embodiment, the subject invention provides a method for selectingone or more ingredients for the composition of the subject invention,wherein the ingredient prevents and/or inhibits the formation of biofilmby a pathogenic microorganism of interest, wherein the method comprisesthe following steps:

a) providing a candidate ingredient or a mixture of candidateingredients, wherein the candidate ingredient(s) is selected from thegroup consisting of microorganisms, extracts of microorganisms, chemicalsubstituents, cellular or acellular components, metabolites ofmicroorganisms, honey, hive products, biosurfactants, prebiotics, plantextracts, and vitamin D;

b) contacting the candidate ingredient(s) with a pathogenicmicroorganism of interest; and

c) selecting a candidate ingredient(s) if said ingredient(s) prevents orinhibits the formation of biofilm by the pathogenic microorganism ofinterest.

In one embodiment, which tests for prevention of biofilm, the pathogenicmicroorganism of interest is in a planktonic state.

In a further embodiment, one or more candidate ingredients areincubated, at a range of concentrations and/or pH, with the pathogenicmicroorganism of interest to determine the optimal concentration and/orpH that prevents or inhibits biofilm formation.

In a further embodiment, the method comprises the step of determiningthe minimum biofilm inhibitory concentration of the selected ingredient,the selected mixture of ingredients, and/or the composition of thesubject invention.

In one embodiment, the candidate ingredient is selected from the groupconsisting of probiotic microorganisms, extract of probioticmicroorganisms, chemical substituents, cellular or acellular components,and metabolites of probiotic microorganisms.

In one embodiment, the probiotic microorganism is selected from thegroup consisting of Aerococcus, E. coli, Bacillus, Enterococcus,Fusobacterium, Lactococcus, Leuconostoc, Melissacoccus, Micrococcus,Oenococcus, Sporolactobacillus, Streptococcus, Staphylococcus,Saccharomyces, Pediococcus, Peptostreptococcus, Proprionebacterium, andWeissella. In another embodiment, the microorganism is selected fromTable 1.

In preferred embodiments, the pathogenic microorganisms or biofilms areselected from methicillin resistant Staphylococcus aureus (MRSA),Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa,Pseudomonas, Haemophilus influenza, Corynebacterium, Candida, orAspergillus.

Diagnosis and Treatment of Diseases Associated with Biofilm Infections

In one embodiment, the subject invention provides methods for preventionand/or treatment of diseases caused by, or associated with, biofilms. Inone embodiment, the method comprises administering, to a subject in needof such treatment, an effective amount of a composition of the subjectinvention.

The term “treatment” or any grammatical variation thereof (e.g., treat,treating, and treatment etc.), as used herein, includes but is notlimited to, ameliorating or alleviating a symptom of a disease orcondition, reducing, suppressing, inhibiting, lessening, or affectingthe progression, severity, and/or scope of a condition.

The term “prevention” or any grammatical variation thereof (e.g.,prevent, preventing, and prevention etc.), as used herein, includes butis not limited to, delaying the onset of symptoms, preventing relapse toa disease, increasing latency between symptomatic episodes, or acombination thereof. Prevention, as used herein, does not require thecomplete absence of symptoms.

The term “effective amount,” as used herein, refers to an amount that iscapable of preventing, ameliorating, and/or treating a pathologicalcondition associated with biofilm.

In one embodiment, “a subject in need of such treatment” refers to asubject who is diagnosed with a pathological condition associated with abiofilm. In a specific embodiment, the subject invention comprisesdiagnosing whether a subject has a biofilm infection, wherein thecompositions of the subject invention are then administered to thesubject who is diagnosed with biofilm infection.

Diagnosis of biofilm infections can be accomplished by clinicaltechniques described in, for example, U.S. Patent ApplicationPublication No. 2010/0285496. The location of pathogenic biofilminfection can be determined by imaging techniques such as, for example,X-ray and CT scans.

In one embodiment, biofilm infection can be detected by:

a) obtaining a biological sample from a subject; and

b) measuring the presence of one or more biomarkers (e.g., proteins,mRNA) that are selectively expressed by microorganisms in a biofilmstate, but not in a free-floating (planktonic) state.

Additionally, biofilm infection can be detected by measuring thepresence of one or more biomarkers that are expressed in elevated levelsby microorganisms in a biofilm state, as compared to levels in afree-floating (planktonic) state. In another embodiment, biofilminfection can be detected by the presence of bacterial extracellularpolysaccharide (EPS) matrix, or chemicals contained in the EPS.

Further, species of pathogenic microorganisms that form biofilm can bedetermined by, for example, using antibodies that recognize antigens orpeptides released by the pathogenic microorganisms, or using probes thatrecognize nucleic acid molecules of the pathogenic microorganisms.

The term “biological sample,” as used herein, includes but is notlimited to, a sample containing tissues, cells, and/or biological fluidsisolated from a subject. Examples of biological samples include but, arenot limited to, tissues, cells, biopsies, blood, lymph, serum, plasma,urine, saliva, and tears. In certain specific embodiments, thebiological samples include tears, nasal fluid, and saliva.

The presence and/or level of biomarkers useful according to the subjectinvention can be determined by techniques known in the art, such as forexample, enzyme-linked immunosorbant assays (ELISA), Western blot,Northern Blot, immunological assays, immunofluorescence, and nucleicacid hybridization techniques.

Diseases Associated with Biofilm Infection

In certain embodiments, the subject invention can be used to prevent,treat, or ameliorate diseases caused by or associated with biofilminfection including, but not limited to, dermatitis, acne, chronicbronchitis, cystic fibrosis, chronic gingivitis, chronic inflammatorybowel disease, chronic eczema, chronic non-healing wounds, chroniccystitis, and medical device related inflammation such as contactlenses. The present inventors also discovered that biofilm infectioncauses or is associated with diseases, such as for example, chronicblepharitis and other chronic inflammatory conditions of the ocular,peri-ocular and dermatologic epithelia such as dry eye syndrome,meibomianitis and rosacea.

In one embodiment, the subject invention can be used to prevent, treat,or ameliorate conditions in otolaryngology practice implicated bybiofilms, including otitis media, chronic sinusitis, chronictonsillitis, adenoiditis, and cochlear and middle ear implant devicefailures. Despite the need for improved treatment methods, prior artmethods such as mechanical disruption (i.e., removal or surgicalexcision of the infected material) or long-term antibiotic treatmentremains the treatment mainstay for chronic inflammatory states due tobiofilm.

The present inventors discovered that certain ocular and peri-ocularinfections result from biofilm-associated chronic inflammatory states.For example, in the ophthalmic field, the presence of biofilms has beenreported on endophthalmitis after cataract surgery, on scleral bucklesafter retinal detachment surgery, punctal plugs, artificial nasolacrimalduct tubing and on soft contact lenses associated with keratitis. Infact, microbial contamination occurs in up to 81% of all contact lenscases, 50% of contact lenses and as 30% of all types of contact lenssolutions, despite use of biocides. Infections associated with bacterialbiofilm formation tend to be persistent, and the most frequentlyisolated organisms from biofilms are Staphylococcus aureus, S.epidermidis, and Pseudomonas aeruginosa. The ocular surfaces of dry eyesand lid margins in chronic blepharitis and contact lens wearers arecolonized by significantly more bacteria and significantly more gramnegative type bacteria than the typically gram positive commensalbacteria found in normal eyes.

In one embodiment, the subject invention can be used to prevent, treat,or ameliorate Chronic rhinosinusitis, another example of a chronicinflammatory state associated with pathogenic biofilm formation.Pathophysiology of chronic rhinosinusitis is likely to involve thedisruption of the normal commensal bacterial population by pathogensfollowed by pathogenic biofilm formation. Typical resulting symptomsinclude nasal dripping, sinus pressure, recurrent headache, post-nasaldrip and cough.

In certain embodiments, the subject invention can be used to prevent,treat, or ameliorate diseases caused by or associated with biofilminfection including, but not limited to, asthma, aspergillosis,“swimmer's ear,” otitis externa, chronic otitis, atopic dermatitis,chronic rhinosinusitis, allergic rhinitis, allergic conjunctivitis,chronic bronchitis, chronic gingivitis, chronic sinusitis, and chronicperiodontitis.

Therapeutic Compositions and Formulations

The subject invention also provides for therapeutic or pharmaceuticalcompositions comprising the ingredients of the invention in a form thatcan be combined with a pharmaceutically acceptable carrier. In oneembodiment, the composition of the subject invention is formulated forocular, periocular, nasal, dental, or pulmonary administration.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum oil such as mineral oil, vegetable oil such as peanut oil,soybean oil, and sesame oil, animal oil, or oil of synthetic origin.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol and the like. The therapeuticcomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsion, creams, lotions, drops,sprays, gel, oils, aerosol, powders, ointment, sustained-releaseformulations and the like. The composition can be formulated withtraditional binders and carriers such as triglycerides. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions contain atherapeutically effective amount of the therapeutic composition,together with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration.

In certain embodiments, the compositions of the subject inventioninclude one or more anti-microbial, anti-bacterial, anti-viral,anti-fungal, or anti-yeast agents.

The therapeutic or pharmaceutical compositions of the invention can beformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude salts formed with hydrochloric, phosphoric, acetic, oxalic, andtartaric acids; and sodium, potassium, ammonium, calcium, and ferrichydroxides, etc.

The subject invention also provides for the modification of theingredient such that it is more stable once administered to a subject,i.e., once administered it has a longer time period of effectiveness ascompared to the unmodified form. Such modifications are well known tothose of skill in the art, e.g., microencapsulation, etc.

Modes of Administration

In one embodiment, a composition of the subject invention is delivered,via local administration, to biological surfaces including, but notlimited to, eyes, teeth, gums, ears, and skin; and non-biologicalsurfaces such as medical devices including, but not limited to,catheters, orthopedic devices, implants, prosthetic heart valves,prosthetic joints, orthopedic implants, shunts, pacemakers anddefibrillators, endotracheal tubes, hemodialysis/peritoneal dialysisdevices, dental implants, and intravascular catheters.

The amount of the therapeutic or pharmaceutical composition of theinvention which is effective in the treatment of a particular disease,condition or disorder will depend on the route of administration, andthe seriousness of the disease, condition or disorder, and should bedecided according to the judgment of the practitioner and each patient'scircumstances. In general, the dosage ranges from about 0.001 mg/kg toabout 3 g/kg. Such a unit dose may be administered more than once a day,e.g. two or three times a day.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary, depending on thetype of the condition and the subject to be treated. In general, atherapeutic composition contains from about 5% to about 95% activeingredient (w/w). More specifically, a therapeutic composition containsfrom about 20% (w/w) to about 80% or about 30% to about 70% activeingredient (w/w).

Following is a detailed description of various components of theinvention.

Probiotics

Probiotics are micro-organisms proving beneficial in some manner to thehuman body. A 2001 World Health Organization symposium on probioticmicro-organisms defined these organisms as “a living micro-organismwhich, when it is consumed in an appropriate amount, has a positiveeffect on the health of its host” (World Health Organization, JointFAO/WHO Expert Consultation on Evaluation of Health and NutritionalProperties of Probiotics in Food Including Powder Milk with Live LacticAcid Bacteria, October 2001). Notably, however, the probioticorganism(s) employable for use in this invention may not be living;moreover, they may, in fact, include micro-organisms or fractionsthereof that would normally be considered commensal or even pathogenicto the human host.

One probiotic may be used singly or probiotics can be combined for usein the invention. They can be used in the present invention in viable ornon-viable form, as a fraction or fractions of the micro-organism(s), asmetabolites of the micro-organisms, inactivated, semi-inactivated orcombinations thereof. Fractions of the biofilm extracellular polymericsubstance (EPS), or biofilm “slime”, may also be used. These variousextracts (fractions, metabolites) may be cellular or acellular and canbe derived from intracellular or extracellular sources. For instance,metabolites such as biosurfactants may be isolated and purified and/ormay be used as is available and contained in micro-organism culturesupernatants. An extract composed of an acellular fraction demonstratingbiosurfactant activity may be used. Such an extract may be used atconcentrations ranging from 10 mcg/ml to 10 grams/ml.

As another example, these extracts may include fractions of biofilmsthat are created when probiotics are cultured in colonies. Suchfractions may be isolated, concentrated, enriched and/or consist ofspent medium. They may be harvested from planktonic cultures and/orbiofilm reactors. They may be harvested at any point in the organisms'growth curves. Alternatively, they may be taken at certain maximal orminimal activity phases of certain enzymes, end-products, etc.

It is well established that probiotics and other microbes/organisms canbe cultured in colonies by methods including but not limited to: trickleflow/wet-dry reactors, high flow reactors, fixed bed reactors, expandedbed reactors, fluidized bed reactors, membrane reactors and spinningdisk reactors. See, for example, Cheng K et al, “Advances in biofilmreactors for production of value-added products”, Appl MicrobiolBiotechnol, 2010, 87:445-456, which is incorporated by reference in itsentirety. The particular methods of doing so are well known to thoseskilled in the art and are readily found in the literature. See, forexample, Cotter, J et al, “Characterization of a modified rotating diskreactor for the cultivation of Staphylococcus epidermis biofilm”,Journal of Applied Microbiology, 2010, 109, 2105-2117; Jackson G et al,“Growing reproducible biofilm with respect to structure and cellcounts”, Journal of Microbiological Methods, 2001, October, 47 (1):1-10; O'Toole, G et al, “Initiation of biofilm formation in Pseudomonasfluoroscens WCS365 proceeds via multiple, convergent signallingpathways: a genetic analysis”, Molecular Microbiology, 1998, 28(3),449-446 and Wu, J et al, “Evaluation of Different Methods for ExtractingExtracellular DNA from the Biofilm Matrix”, Applied and EnvironmentalMicrobiology, August 2009, p. 5390-5395; all references are hereinincorporated by reference in their entirety.

It is also well known that biofilms can be fractionated by manydifferent methods, including but not limited to the techniques ofcentrifugation, filtration, heating, blending, sonication, treatmentwith complexing agents, treatment with ion (cation) exchanger resins andsodium hydroxide (Nielsen, P H and Jahn, A, Microbial ExtracellularPolymeric Substances (eds Wingender, J., Neu, T., Flemming, H. C.), p.49-72, Springer-Heidelberg, 1999; Thomas D P et al, “Proteomics for theanalysis of the Candida albicans biofilm lifestyle”, Proteomics, 2006,6(21); 5795-804, Flemming, H et al, “The biofilm matrix”, Nature Reviewsin Microbiology, 2010, 8(9): 623-633; all references herein incorporatedby reference in their entireties).

Fractions of biofilms such as exopolysaccharides can be concentratedthrough precipitation with alcohols such as ethanol or acetone (Kanmani,P et al, “Production and purification of a novel exopolysaccharide fromlactic acid bacterium Streptococcus phocae P180 and its functionalcharacteristics activity in vitro”, Bioresource Technology, 2011,article in press; Aguilera, A et al, “Extraction of extracellularpolymeric substances from extreme acidic microbial biofilms”, ApplMicrobiol Biotechnol, 2008, 78(6):1079-1088; all references are hereinincorporated by reference in their entirety). Such an extract could alsobe used as described in TABLE 6 in concentrations ranging from 1 mg/mlto 1000 mg/ml.

Specific examples of probiotics suitable for use in the presentinvention include non-lactic acid as well as lactic acid producingbacteria (LAB). These include the species Bacteroides, Bifidobacterium,and Lactobacillus; also, certain strains of Aerococcus, E. coli,Bacillus, Enterococcus, Fusobacterium, Lactococcus, Leuconostoc,Melissacoccus, Micrococcus, Oenococcus, Sporolactobacillus,Streptococcus, Staphylococcus, Saccharomyces, Pediococcus,Peptostreptococcus, Proprionebacterium, and Weissella. Examples ofmicro-organisms suitable for use in the invention have been listed anddescribed more extensively elsewhere (see Table 1). In one instance, forexample, an extract of Lactobacillus acidophilus (ATCC 4356) and/or anextract of Escherischia coli K12 (ATCC 10798) and/or Streptococcusthermophilus 4022 (such as ATCC 19258) strains may be used.

The anti-inflammatory activity of the invention is most prominent atcell concentrations ranging from 10 million to 10 billion colony-formingunits (CFUs) per milliliter (mL) (and/or the amount of metabolitesproduced by these numbers of bacteria).

TABLE 1 EXEMPLARY PROBIOTIC ORGANISMS AND COMMERCIAL SOURCES IdentifierStrain Commercial Source Bacillus coagulans Ganeden Inc., USA ATCC 25527Bacteroides adolescentis CNCM I-2168 Bifidobacterium animalis ATCC(American Tissue Type Collection, Manassas, VA) B. bifidum ATCC 15700 B.breve ATCC DPTC 001 B. breve R-070 Institut Rosell Inc., Montreal,Quebec, Canada ATCC 15697 B. infantis ATCC DPTC 047 B. infantis BBI Chr.Hansen, Milwaukee, WI DPTC 002 B. lactis Bb12 (ATCC27536) Chr. Hansen B.lactis NCC2818 (CNCMI-3446) ATCC 15708 B. longum ATCC DPTC 004 B. longumBB46 Chr. Hansen DPTC 003 B. longum BBL Chr. Hansen B. longum NCC490(CNCMI-2170) (a.k.a., B. longum Bb536 or Morinaga strain) DPTC 036 B.spp. Rolly fermented milk, Snow Brand Escherischia coli M-17 BioBalanceInc., USA E. coli K12 E. coli Nissle ATCC 4356 Lactobacillus acidophilusATCC ATCC 700396 L. acidophilus ATCC DPTC 025 L. acidophilus Mil Milfermented milk, Yakult, Tokyo, Japan DPTC 049 L. acidophilus Mil Milfermented milk, Yakult DPTC 046 L. acidophilus AS-1 Quest International,Rochester, MN DPTC 027 L. acidophilus DDS-1 Capsule supp., Natren Inc.,Westlake Village, CA DPTC 010 L. acidophilus HP10 NortheastNutraceuticals, S. Boston, MA DPTC 011 L. acidophilus HP100 NortheastNutraceuticals DPTC 012 L. acidophilus HP101 Northeast NutraceuticalsDPTC 013 L. acidophilus HP102 Northeast Nutraceuticals DPTC 014 L.acidophilus HP103 Northeast Nutraceuticals DPTC 015 L. acidophilus HP104Northeast Nutraceuticals DPTC 048 L. acidophilus HP15 NortheastNutraceuticals DPTC 005 L. acidophilus NCFM Rhodia Inc., Madison, WIDPTC 006 L. acidophilus NCFM North Carolina State University, Raleigh,NC DPTC 007 L. acidophilus PIM703 Chr. Hansen DPTC 008 L. acidophilusSBT2062 Snow Yogurt + 2, Snow Brand L. alimentarius ATCC 33620 L.amylovorus ATCC ATCC 393 L. casei ATCC DPTC 051 L. casei DN-114 001Actimel Original fermented milk, Danone, Paris, France DPTC 034 L. caseiLC10 Rhodia DPTC 035 L. casei PIM661 Chr. Hansen DPTC 033 L. caseiShirota Joie fermented milk drink, Yakult DPTC 030 L. casei ShirotaHealth drink produced by Yakult ATCC 33820 L. crispatus ATCC DPTC 009 L.crispatus BG2FO4 NCSU L. curvatus ATCC 11842 L. delbrueckii ssp.bulgaricus ATCC DPTC 020 L. delbrueckii ssp. bulgaricus 2038 Yogurt,Meiji Milk Products Co. Ltd., Tokyo, Japan DPTC 021 L. delbrueckii ssp.bulgaricus 2038 Yogurt, Meiji DPTC 019 L. delbrueckii ssp. bulgaricusMR120 Rhodia DPTC 022 L. delbrueckii ssp. bulgaricus PIM695 Chr. HansenL. delbrueckii ssp. lactis DPTC 045 L. rhamnosus MX1 University ofWestern Ontario, London, Ontario, Canada ATCC 33199 L. gallinarum ATCCATCC 33233 L. gasseri ATCC DPTC 026 L. gasseri ADH NCSU DPTC 016 L.helveticus MR220 Rhodia DPTC 017 L. helveticus NCK388 NCSU ATCC 33200 L.johnsonii ATCC DPTC 028 L. johnsonii 11088 (NCK 088) NCSU DPTC 029 L.johnsonii La-1 Nestle', Lausanne, Switzerland L. johnsonii CNCM I-1225DPTC 018 L. lactis San Chr. Hansen ATCC 25302 L. paracasei ATCC L.paracasei Lpc-37 L. paracasei ST11 NCC 2461 (a.k.a., CNCM I-2116) ATCC23272 L. reuteri ATCC DPTC 037 L. reuteri 1063-S Biogaia Biologics,Stockholm, Sweden DPTC 038 L. reuteri 11284 Biogaia Biologics DPTC 039L. reuteri SD2112 Biogaia Biologics DPTC 040 L. reuteri T-1 BiogaiaBiologics ATCC 7469 L. rhamnosus ATCC DPTC 042 L. rhamnosus GR-1University of Western Ontario DPTC 043 L. rhamnosus R-011 InstitutRosell DPTC 044 L. rhamnosus R-049 Institut Rosell ATCC 53103 L.rhamnosus GG ATCC Lactococcus lactis Leuconostoc mesenteroides,subspecies cremoris Proprionibacterium freudenrichii, subspeciesshermanii JS ATCC 10556 Streptococcus salivarius S. mitis S. oralis S.sanguis S. thermophilus S244 ATCC Staphylcoccus carnosus S. xylosusVitreoscilla filiformis Yeast Lyophilized yeast extract Centro RicercheYOMO, Milan Saccharomyces cerevisiae Health Sciences USA ATCC 74012 S.boulardii Biocodex, Gentilly, France; ATCC ATCC MYA-797 S. boulardiiATCC S. subtilis

Exemplary Materials and Methods Relating to Culture and Fractionation ofMicro-Organisms

As already discussed, the invention uses various materials and methodspertaining to bacterial and non-bacterial micro-organism and biofilmgrowth and harvesting. For example, filtrates from the yeastSaccharomyces may be obtained using protocols such as those described byKrasowska A et al, “The antagonistic effect of Saccharomyces boulardiion Candida albicans filamentation, adhesion and biofilm formation”, FEMSYeast Res ((2009) 1312-1321. Fractionation of planktonic and biofilmbacterial and non-bacterial cultures into various subcompartments suchas nuclear, subnuclear, cytoplasmic, lysate, supernatant, spent medium,cellular membrane, biosurfactant, extracellular DNA, extracellular RNAand so on may then occur using any of the techniques previouslyreferenced.

In particular, biosurfactants produced or yielded from micro-organismscan be obtained, isolated and enriched using a number of methods. Theseare described in scientific literature such as Baker S C et al,“Enrichment and purification of lipopeptide biosurfactants”, Adv Exp MedBiol 2010; 672:281-288, Rivardo, F et al, “Anti-adhesion activity of twobiosurfactants produced by Bacillus spp. prevents biofilm formation ofhuman bacterial pathogens”, Appl Microbiol Biotechnol (2009) 83:541-553,Gudina, E et al, “Isolation and functional characterization of abiosurfactant produced by Lactobacillus paracasei”, Colloids andSurfaces B: Biointerfaces 76 (2010) 298-304, Gudina E et al,“Antimicrobial and antiadhesive properties of a biosurfactant isolatedfrom Lactobacillus paracasei ssp. paracasei A20”, Letters in AppliedMicrobiology, 50 (2010) 419-424, Sarachat, T et al, “Purification andconcentration of a rhamnolipid biosurfactant produced by Pseudomonasaeruginosa SP4 using foam fractionation”, Bioresource Technology 101(2010) 324-330 and Rivardo F et al, “Synergistic effect of lipopeptidebiosurfactant with antibiotics against Escherischia coli CFT073biofilm”, International Journal of Antimicrobial Journal, (2011),article in press. All publications are herein incorporated by referencein their entirety.

Additionally, screening methods may be used to identify and assessbiosurfactant producing micro-organisms. These techniques are readilyfound in the literature such as those described in Burch A et al, “NovelHigh-Throughput Detection Method To Assess Bacterial SurfactantProduction”, Applied and Environmental Microbiology, August 2010, p.5363-5372 and Walter V et al, “Screening concepts for the isolation ofbiosurfactant producing micro-organisms”, Adv Exp Med Biol 2010;672:1-13. These publications are herein incorporated in their entiretyby reference.

Honey and Other Hive Products

Used for centuries in the treatment of various ailments includinginfection, honey has been shown to have antimicrobial as well asanti-inflammatory effects (Viuda-Martos, M, “Functional Properties ofhoney, propolis and royal jelly”, J Food Sci 2008 November;73(9):R117-24). Reasons for these effects include a polysaccharidecalled methyl glyoxal, MGO, (Adams, C, “The origin of methylglyoxal inNew Zealand manuka (Leptospermum scoparium) honey”, CarbohydrateResearch, 2009 May 26; 344(8):1050-1053), hydrogen peroxide activity,low pH, high osmolarity and antimicrobial peptide activity (Kwakman, P,“How honey kills bacteria”, FASEB J, 2010 July; 24(7):2576-82). Certainhoney has been shown to have anti-biofilm effect against a fewpathogens; but there are no commercially available honey-based productsindicated for the treatment, removal, or prevention of disease-relatedbiofilms.

The present invention also provides methods of manufacture andcharacterization as well as the clinical use of standardized,pharmaceutical-grade honey. A very common ingredient of the invention atthis time includes medical grade honey, such as Medi-Honey (ComvitaInc., New Zealand), which has been sterilized for use on wounds. As theantiseptic quality of honey varies based on time of year, pollens andnectars gathered, lot, season, etc., each batch of manuka honey has ameasurement taken of its antiseptic potency called the “Unique ManukaFactor.” As previously discussed, the UMF® number comes from alaboratory test for antibacterial activity, with the honey beingcompared against a standard reference antiseptic, phenol. For instance,UMF® 20+ would be equivalent in antiseptic potency to a 20% solution ofphenol. No other honey to date has antimicrobial qualities measured thisway.

However, this method of antiseptic characterization has 2 significantdisadvantages—antimicrobial activity against the micro-organism to betreated is unknown, and this measurement is applicable only to manukahoney, and no other honey. All honey has anti-microbial qualities, whichare very likely to be variable from type to type. If honey is to beincluded as part of the pharmaceutical anti-microbial armamentarium, itneeds to be characterized like antibiotics are, via standardized,validated tests. For today's antibiotics, these include bacterial andfungal inhibitory concentration (MIC) tests as documented in theNational Committee on Clinical Standards (NCCLS) reference manual(herein incorporated by reference). Anti-viral activity of honey mayalso be determined for particular viruses such as Respiratory SyncytialVirus (RSV) via certain procedures such as that described by Sudo K,“YM-53403, a unique anti-respiratory syncytial virus agent with a novelmechanism of action”, J of Antiviral Research, 2005 (65): 125-131,herein incorporated as reference. Other well-accepted tests foranti-viral activity available commercially which may be used include theOxoid M.I.C.Evaluators (Oxoid Ltd., United Kingdon), the Etest(BioMerieux, France) and similar others.

In particular, the anti-biofilm activity of honey needs to bestandardized via biofilm inhibitory concentration (BIC) tests, which maybe done, for example, by subjecting samples to the Minimum BiofilmElimination Concentration (MBEC) test procedure (see U.S. Pat. No.6,051,423 by Ceri et al, which is herein incorporated by reference).

As is done with antibiotics in MIC testing per standard NCCLS protocol,BIC tests of honey should be performed against common human pathogenssuch as S. aureus, Pseudomonas, methicillin resistant and methicillinsensitive Staphylococcus aureus, Haemophilus influenza, Corynebacterium,Candida, Aspergillus, etc. An alternative means of anti-biofilm efficacytesting using characterized biofilm spheres is also described herein.Due to intrinsic variability, each lot from various monofloral sourcesutilized in foraging by the honey bees (manuka, pine, orange, and so on)will be characterized according to the anti-microbial/anti-biofilmactivity of the resultant honey.

Specific anti-microbial/anti-biofilm activity from monofloral honey willthen be compared to various antibiotics, i.e., orange blossom honeyversus ampicillin in terms of activity against Staphylococcus aureus.This is particularly important in the case of methicillin resistantStaphylococcus aureus. Alternatively, monofloral honey may be generatedthrough the use of enclosed greenhouses in which one pollinating planttype is grown and honey bees are kept. Honey is then harvested fromthese hives, ensuring their monofloral and consistent nature.

Honey meant for medical use would then be sterilized via low-dose gammairradiation or micro-filtration to be used as a topical equivalent foranti-microbial purposes as previously described in the presentinvention. Honey may be used between 10 and 90% volume/volume (hiveproduct/invention). Various monofloral honeys could also be combined toprovide better, more broad-spectrum coverage.

In reference to honey or hive products, samples are diluted in sterile,pH-neutral normal saline solution to test concentrations varying from0.001% to 100% and used in place of the antibiotic(s) named in theprotocols referenced above.

Hive products other than honey may also be used. For example, propolisand royal jelly, ranging from 1 mcg/ml to 10 mg/ml, may be used,prepared via methods described below.

During preparation of the invention, various samples comprised ofprobiotic extract and honey (e.g., Lactobacillus and manuka honey) maybe processed using various methods and/or may remain unprocessed.Processing may include cell fractionation, heat treatment,ultrasonication, filtration, enzymatic treatment, gamma irradiation orother means of sterilization, micronization, biosurfactant isolation,crystallization and/or lyophilization, of extract samples showinganti-biofilm and anti-inflammatory activities.

Exemplary Materials and Methods Relating to Honey and Other HiveProducts Preparation of Various Extracts of Sterile Hive Products

During preparation of the invention, various samples comprised of hiveproducts such as honey, propolis, royal jelly, bee bread or bee pollenmay be processed using various methods and/or may remain unprocessed.Processing may include fractionation of proteins, sugars, pollens,polyphenols or other constituents via chemical means, high performanceliquid chromatography, physical (filter) ultrafiltration, gelelectrophoresis, heat treatment, enzymatic treatment, micronization,ultrasonication, crystallization, dehydration or lyophilization.

For instance, fractionation may be performed as described inSalazar-Olivo, L. A. et al, “Screening of biological activities presentin honeybee (Apis mellifera) royal jelly”, Toxicology in Vitro 19 (2005)645-651; Mishima S et al, “Effects of propolis on cell growth and geneexpression in HL-60 cells”, J Ethnopharmacol 2005, 99:5-11; Nakajima Yet al, “Comparison of bee products based on assays of antioxidantcapacities”, BMC Complementary and Alternative Medicine 2009, 9:4;Santos, F. et al, “Antibacterial activity of Brazilian propolis andfractions against oral anaerobe bacteria”, J Ethnopharmacol 2002, 80:1-7and Maruyama, H et al, “Anti-inflammatory effect of bee pollen ethanolextract from Cistus sp. of Spanish origin on carrageenan-induced rathind paw edema”, BMC Complementary and Alternative Medicine 2010, 10:30,Yu, F et al, “Royal Jelly Proteome Comparison Between A. melliferaligustica and A. cerana cerana”, Journal of Proteome Research 2010, 9,2207-2215 and Scarselli, R et al, “Towards royal jelly proteome”,Proteomics 2005, 5, 769-776.

Biosurfactants

Biosurfactants are compounds released by microorganisms, and aregenerally non-toxic and biodegradable. In one embodiment, biosurfactantsuseful according to the subject invention are released by probioticsincluding non-lactic acid and lactic acid producing bacteria (LAB). Inone embodiment, biosurfactants useful according to the subject inventionare released by probiotics including, but not limited to, Bacteroides,Bifidobacterium, and Lactobacillus.

In additional embodiments, biosurfactants can be released by certainstrains of Aerococcus, E. coli, Bacillus, Enterococcus, Fusobacterium,Lactococcus, Leuconostoc, Melissacoccus, Micrococcus, Oenococcus,Sporolactobacillus, Streptococcus, Staphylococcus, Saccharomyces,Pediococcus, Peptostreptococcus, Proprionebacterium, or Weissella. Inanother embodiment, biosurfactants can be released by one or moreorganisms listed in Table 1.

Biosurfactants useful according to the subject invention can beglycolipids or lipoproteins. In one embodiment, the biosurfactants canbe glycolipids, lipopeptides, depsipeptides, phospholipids, substitutedfatty acids, lipopolysaccharides, surlactin, surfactin, visconsin,spiculisporic acid, or rhamnolipids.

Prebiotics

Prebiotics are nondigestible, fibrous fructo- orgalacto-oligosaccharides (FOS or GOS) found in many plants that aremetabolized by the large intestine to form short chain fatty acids suchas butyrate. These fatty acids metabolically support probiotic coloniesin the intestine, as well as help generate an effective local innateimmune response. Consequently, prebiotic supplementation may increaseefficacy of probiotic supplementation. This combination is known assynbiotic therapy.

In certain embodiments, the invention may make use of certainprebiotics, such as locust-bean (carob) gum, in the concentrationbetween 10 mcg-100 mg per milliliter, to augment anti-biofilm efficacy.These include fructo-oligosaccharides (FOS), manno-oligosaccharides(MOS), galacto-oligosaccharides (GOS), arabinogalactans and otherdietary fibers, inulin, lactulose, resistant starch, isomalt, oat bran,and pectin. Larch arabinogalactan may be used and is also known as AG,Ara-6, Arabinogalactan, Arabinogalactin, dietary fiber, larch, larchgum, larch tree, larix, Mongolian Larch, Mongolian Larchwood, Solublefiber, Stractan, Western Larch, Western Larch Arabinogalactan, Wood Gum,Wood Sugar, Larix decidua, Larix europaea, Pinus Larix, Larixoccidentalis, Larix gmelinii var. gmerlinii, Larix dahurica, and Abiesgmelinii. Also may be used: konjac glucomannan, also known as konjacgum, hydrolyzed konjac, hydrolyzed glucomannan, unhydrolyzed konjac,hydrolyzed glucomannan, Manna, Konjac, Konjac fiber, Devil's Tongue, andElephant-Foot Yam. Also may be used: soluble or insoluble beta glucan,also known as the bran of cereal grains, plant cellulose, fungalcomponents, mushroom components, seaweed components, curdlan, laminarin,chrysolaminarin, lentinan, Polysaccharide-K, lichenin, pleuran, xanthanand zymosan.

Plant Extracts

Plant extracts are known to have anti-inflammatory and anti-microbialproperties. Plant extracts used in some embodiments of the inventioninclude horseheal (Inula helenium, L. Asteraceae, elecampane), rose(Rosa damascena L., Rosaceae), lavender (Lavandula angustifolia L.,Labiatae), chamomile (Matricaria recutica L., Asteraceae), orange(Rutaceae), eucalyptus (Eucalyptus globulus L., Myrtaceae), geranium(Geranium robertianum L., Geraniaceae), juniper (Juniperus communis L.,Cupressaceae), citrus (Citrus sinensis L., Rutaceae), tea tree(Melaceuca alternifolia), manuka bush (Leptospermum scoparium), neemtree (Azadirachta indica, A. Juss), tea plant (Camellia sinensis) androsemary oils (Rosmarinus officinalis L., Lamiaceae). Essential oil orwater distillate of the above botanicals may be used. For instance,manuka oil at a concentration between 1-10% volume/volume (plantextract/invention) may be used. Plant materials, distillates and plantoils of pharmaceutical grade quality may be purchased directly fromsuppliers or a hydrodistillate essence may also be generated per methoddescribed below which may also be used at a concentration between 1-10%volume/volume (plant extract/invention).

Exemplary Materials and Methods Relating to Plant Extracts PreparationMethod for Plant Extract Using Clevenger Type Apparatus or SimilarTechniques

During preparation of the invention, plant extracts may be prepared invarious ways, particularly regarding purification of their essentialoils. Please refer to Clevenger, J F, “Apparatus for the determinationof volatile oil”, 1928; J Am Pharm Assoc, 17; 346, incorporated hereinin its entirety by reference. Other techniques may be used such as thosedescribed in Vian, M et al, “Microwave hydrodiffusion and gravity, a newtechnique for extraction of essential oils”, Journal of ChromatographyA, 1190 (2008), 14-17 or Farhat, A et al, “Eco-friendly and cleanerprocess for isolation of essential oil using microwaveenergy—experimental and theoretical study”, Journal of Chromatography A,1216 (2009), 5077-5085.

Other Plant Extracts—Camellia sinensis Extracts

The invention also makes use of tea (Camellia sinensis) derivatives.More specifically, L-theanine and/or green tea polyphenols such asepigallocatechin gallate (EGCG), each in the amounts of 10 mcg/ml to 10mg/ml, may be used.

Alkylamine antigens are known to stimulate innate immune response. Themost concentrated plant source of alkylamine antigen is an amino acid ingreen tea known as L-theanine (Bukowski, J et al, “Human gamma delta Tcells recognize alkylamines derived from microbes, edible plants andtea:implications for innate immunity”, 1999, Immunity, Vol. 11, 57-65).L-theanine is represented by the chemical formulaC2H5NHCOCH2CH2CH(NH2)COOH, D- and or L,D-theanine,2-Amino-4-(ethylcarbamoyl)butyric acid, N-ethyl-L-glutamine,gamma-glutamylethylamide, N-gamma-Ethyl-L-glutamine,gamma-ethylamino-L-glutaminic acid, Suntheanine, 5-N-ethylglutamine,green or other tea extract, raw or refined tea leaves, roots or othercomponents of Camellia sinensis or certain other Camellia species (i.e.,C. japonica, C. sasanqua) or particular species of mushroom (i.e.,Xerocomus badius).

Some tea polyphenols are known to have anti-oxidant and anti-microbialeffects. Epigallocatechin gallate (EGCG) is the main polyphenolcomponent of green tea. Other polyphenols include epigallocatechin,epicatechin gallate and epicatechin.

Vitamin D

Vitamin D has recently-discovered effects on the innate immune systembesides its well known effects on bone metabolism. Vitamin D3 inducesproduction of anti-microbial peptides (AMPs) such as cathelicidin (LL37)on body surfaces such as the skin and eye.

Vitamin D3 may be added to the formulation as an additional activeingredient. More specifically, the active form of Vitamin D may be usedin an amount ranging from 1 mcg to 1 mg/ml. Yet more specifically, aform of water soluble Vitamin D currently under patent protection may beused.

Other Viable and Nonviable Organisms

Other viable or nonviable microorganisms which may be used include thosethat can be administered safely to humans, including human commensals,certain organisms found in the environment such as marine organisms orextremophiles such as organisms found in geothermal vents or hot springsand/or their extracts or metabolites. Human commensals arenon-pathogenic organisms which normally colonize the human body whilecausing no disease. These include certain members of the kingdoms Moneraand Fungi, including the phylum Ascomycota.

Also may be used are the yeasts Candida, Saccharomyces, Kluyveromyces,Debaromyces, Zygosaccharomyces and Schizosaccharomyces as well asYarrowia, Torulaspora, Pichea and the molds Aspergillus and Penicillium.Also may be used: baker's yeast cell wall or cell wall components suchas zymosan (Saccharomyces cerevisiae) or other yeasts, fungal componentsor mushroom components. Also may be used: algae, fungal and bacterialextracts ranging from 10 mcg to 100 mg weight/volume (extract/invention)and/or viable or nonviable organisms ranging between 10 million to 10billion colony-forming units (CFUs) per milliliter (mL) (and/or theamount of metabolites produced by these numbers of organisms).

Exemplary Materials and Methods Relating to Other Viable and NonviableMicroorganisms

Polysaccharide Extract Preparation from Spirulina platensis

During preparation of the invention, particular extracts may be preparedfrom other, non-bacterial organisms, including the blue-green alga,Spirulina platensis. In particular, methods as described in Yang, L etal, “Inhibitory effects of polysaccharide extract from Spirulinaplatensis on corneal neovascularization”, Molecular Vision 2009; 15:1951-1961 or Pugh, N et al, “Isolation of three high molecular weightpolysaccharide preparations with potent immunostimulatory activity fromSpirulina platensis, Aphanizomenon flos-aqae and Chlorella pyrenoidosa”,Planta Med 67 (2001) 737-742 may be used.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting.

Example 1—Specific Embodiments for Ocular, Periocular and Sinonasal Use

Specific sites of administration of the compositions of the subjectinvention include, but are not limited to, ocular, nasal, oral and skin.Carriers useful according to the subject invention include, but are notlimited to, spray, cream, ointment, lotion, gel, drop, soap or any otherform appropriate to the site of administration. These compositions canbe prepared using standard methods known to those skilled in the art.

Embodiments of the invention may also be applied to inert objects, forexample, as cleansing agents, such as aerosol and nonaerosol sprays,washes, soaps, etc. Embodiments may be administered as a single use, ormay be administered once or more in a 24 hour period.

Although the following formulations are used for illustration, otherformulation types may be used. Formulations are preferably preparedunder a biocontainment hood using aseptic techniques for maximumsterility. Formulations 1-10 may use Formulae 1-10 as described above asthe active components. However, each individual component may also beadded individually as described below. As in the above Formulae, variousorganisms may be substituted for any strains of the probiotic S.thermophilus.

Formulation 1—Eye Drop Solution for Symptomatic Relief of Dry, IrritatedEyes

2 ml S. thermophilus 100 Billion CFU/1 ml sterile PBS5 ml undiluted manuka honey3 mls sterile normal saline solution10 mls of 20 B CFU/ml 50% honey solution

In techniques well known to practitioners of the art, the preparation isaliquoted into sterile eye-drop bottles and adjusted to neutral pH. Eachbottle of eye drop solution may contain a chosen volume of theformulation, for example, 10 mls in 15 ml eye-drop bottle. The eye dropbottle may be stored at, for example, 4 degrees C. throughout the lifeof its use.

Formulation 2—Eye Cream for the Symptomatic Relief of Dry Eyes andIrritated Eyelids

500 B CFU freeze-dried S. thermophilus5 mls undiluted honey45 mls cold cream base50 mls of 10 B CFU/ml 10% honey in cold cream base

The above ingredients may be mixed in a standard cold cream well knownto practitioners of the art, i.e., one composed of a base of cetylesters wax, white wax, mineral oil, sodium borate and purified water.The preparation may be stored in a sterile jar containing 20 mls of theinvention. The cold cream mixture may be stored at 4 degrees C.throughout the life of its use.

Formulation 3—Nasal Solution for the Symptomatic Relief of ChronicSino-Nasal Congestion

10 mls nonsterile or sterile manuka honey (Medi-Honey, Comvita Inc., NewZealand)39 mls sterile normal saline solution to a final concentration of 20%v/v1 ml 50B CFU/ml PBS freeze-dried inactivated S. thermophilus (ATCCBAA-250)50 mls of 1 B CFU/ml 20% honey in normal saline base

Solutions can be prepared with or without preservatives and/oranti-oxidants and/or viscosity enhancers. Solutions can be filteredthrough 0.2 micron filters (Millipore) into 15, 20 or 30-ml sterilenasal spray bottles. The nasal spray bottle was kept stored at 4 degreesC. throughout the life of its use or alternatively may be kept at roomtemperature for up to 30 days.

Formulation 4—Nasal Irrigation Packets for Symptomatic Relief of ChronicSinus Congestion

500 mg dehydrated manuka honey (see above for procedure).Freeze-dried inactivated S. thermophilus (ATCC BAA-250) 100 billionCFU/ml (using appropriate excipients, if needed) when reconstituted in250 mls of water.

Powdered USP grade blend of sodium chloride and sodium bicarbonateproducing isotonic and pH neutral solution when reconstituted in 250 mlswater. Contents of one nasal rinse packet that when reconstituted in 250mls of water by the consumer results in 375 M CFU/ml in 4 mg/ml honeysolution.

The invention in this form may be packaged with a nasal rinse bottlewith a volume of 250 mls. Alternatively, the packets themselves may bepackaged alone.

Formulation 5—Nasal Irrigation in Concentrated Liquid Form for theSymptomatic Relief of Chronic Sino-Nasal Congestion

2.5 mls manuka honey (Medi-Honey, Comvita Inc., New Zealand)2.0 mls sterile normal saline solution at neutral pH0.5 ml freeze-dried inactivated S. thermophilus (ATCC BAA-250) 100 BCFU/ml in sterile PBS5 mls of 2 B CFU/ml in 1% honey solution (final reconstitution byconsumer)

Solutions may be prepared with or without preservatives and/oranti-oxidants and/or viscosity enhancers. Solutions may be filteredthrough 0.2 micron filters (Millipore) into sterile 10 mls disposablecontainers. The solutions may or may not be packaged with nasal rinsebottles of appropriate volume to reach appropriate tonicity such thatfinal solution when mixed with 250 mls water is isotonic.

Example 2—Specific Embodiments for Cutaneous Use

Formulation 6—Gel for Cleansing of Chronically Irritated Dry and/orNormal Skin50 mls manuka honey, sterile or non-sterile40 mls normal saline solution at neutral pH containing 5%methylcellulose 1500 cP, 10% v/v10 ml freeze-dried inactivated S. thermophilus (ATCC BAA-250) 100 BCFU/ml in sterile PBS100 mls of 10 B CFU/ml in 50% honey solution

The final product may be packaged in sterile hand-pump containers witheach pump dispensing 5 mls of the invention per pump cycle. Thisparticular form may be left on the area applied and does not requirewashing.

Formulation 7—Liquid Soap for Cleansing of Chronically Irritated Dryand/or Normal Skin50 mls glycerin liquid soap base50 mls concentrated liquid form(s) of the invention (i.e., Formulation 5above)100 mls of 1 B CFU/ml in 0.5% honey solution

Above constituents may be combined with gentle heating and stirring andpoured into 100 mls hand pump bottles, cooled and packaged.

Formulation 8—Bath Additive for Cleansing of Chronically Irritated Dryand/or Normal Skin10 grams sodium citrate20 grams sodium bicarbonate10 grams crystallized or dehydrated manuka honey (see above)100 B CFU freeze-dried inactivated S. thermophilus (ATCC BAA-250)5 mls essential oil of lavender5 mls essential oil of manuka50 gm 2 B CFU/gm 20% honey mixture

May be cured with witch hazel, placed into 50 ml domed molds, allowed toharden and packaged in air-tight disposable packaging.

Example 3—Specific Embodiments for Environmental Use

Formulation 9—Environmental Anti-Biofilm Spray for Cleansing ofInanimate Surfaces which May be Exposed to Pathogenic BiofilmColonization80 mls cleansing spray vehicle in dilute alcohol or vinegar base10 mls non sterile manuka honey10 mls 100 B CFU/ml PBS freeze-dried inactivated S. thermophilus ATCCBAA-250100 mls 10 billion CFU/ml 10% honey solution

May be packaged in hand-pump room spray containers; each pump maydispense an aerosol volume equivalent to 1 ml of solution. Thisparticular form may be left on the area applied and does not requirewashing.

Example 4—Specific Embodiments for OTIC Use Formulation 10—Ear Drop forSymptomatic Relief of Chronically Irritated, Inflamed Ears

3 ml gamma irradiated, sterile manuka honey (Medi-Honey, Comvita Inc.,New Zealand)6 mls sterile glycerin1 ml S. thermophilus 100 Billion CFU/1 ml sterile PBS10 mls 10 Billion CFU/ml 30% honey solution

Example 5—Specific Embodiments for Respiratory Use Formulation11—Inhalant Solution

50 mg citric acid, anhydrous2 mls gamma irradiated, sterile manuka honey (Medi-Honey, Comvita Inc.,New Zealand)1 ml 50 Billion CFU/ml S. thermophilus CFU/ml sterile PBS97 mls sterile normal saline100 mls 500 Million CFU/ml S. thermophilus CFU/ml 2% honey solution

Example 6—Specific Embodiments for Dental Use Formulation 12—Toothpaste

15 mls xylitol

3 mg NaCl

38 gr glycerin2 mls thymol distillate (i.e., Monarda genus)20 gr manuka honey1 ml S. thermophilus 100 Billion CFU/1 ml sterile PBS80 mls S. thermophilus 1.25 B CFU/1 ml

Example 7—Specific Embodiments for Periodontal Use Formulation13—Curettage Agent for the Treatment of Periodontitis

Lyophilized S. thermophilus, at final concentration of 10 B CFU/gram ofvehicle.Manuka honey, dehydrated at 120 F, powder-milled; at final concentrationof 50 gr/vehicle.Poly(esters) on polylactide (PLA), polyglycolide (PGA), polycaprolactone(PCL) or other biodegradable copolymers may be used as a deliveryvehicle for this formulation intended for subgingival administrationinto periodontal pockets.

The above formulation may be modified so that it could be used as acoating for dental floss, incorporated into a mouthwash, gum or lozenge.

Prebiotics, plant oil(s) and other components referenced in the detaileddescription of the ingredients may be added to any of the formulationsof the ingredients if desired, such as other organisms including but notlimited to Spirulina, Vitamin D3, L-theanine and EGCG.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

1-20. (canceled)
 21. A liquid probiotic composition for oral consumptioncomprising biologically-active biofilm cellular mass, a liquid ediblematerial, and further comprising exopolysaccharides produced by thebiofilm.
 22. The composition, according to claim 21, wherein thebiological activity of said biofilm cellular mass is anti-microbialactivity and/or anti-inflammatory activity.
 23. The composition,according to claim 21, comprising a biosurfactant produced by thebiofilm.
 24. The composition, according to claim 21, wherein the ediblematerial comprises ingredients that are generally regarded as safe(GRAS).
 25. The composition, according to claim 21, wherein the biofilmcellular mass is a Lactobacillus biofilm cellular mass.